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Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa

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The identification of transcripts that vary during the acquisition of superior tolerance to environmental stress contribute to the understanding of the molecular bases of adaptation to adverse growth conditions. SRAP-cDNA was used to identify genes that were differentially expressed between plants of alfalfa (Medicago sativa L.) that were non-acclimated (NA) or hardened to a non-lethal freezing temperature (HF). Search of polymorphism was performed within the cultivar Apica (ATF0) and population ATF5 obtained after five cycles of recurrent selection for superior tolerance to freezing (TF) within ATF0. Analysis of bulked cDNA from 50 non-acclimated and 50 cold-acclimated plants in each population identified transcripts that vary in abundance in response to acclimation to sub-zero temperature and recurrent selection. Sequencing of purified fragments revealed significant homologies with genes with key physiological functions that are distributed across the Medicago truncatula genome. Regulatory genes including receptor-like kinases, a phytochrome-interacting factor and a phosphoinositide phosphatase are among potential candidates involved in the improvement of freezing tolerance by RS. RT-qPCR analysis of samples from populations recurrently selected within the cultivars Apica and Evolution confirmed the common cold-induced responses in unrelated genetic backgrounds. Our results show that random amplification of cDNA in combination with bulk segregant analysis of pooled samples from recurrently selected populations is an effective strategy to identify functional sequences putatively associated with superior acclimation to environmental stress in open-pollinated species.

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Funding was supported by Competitive grant program of Agriculture and Agri-Food Canada (Grant No. Project 1759).

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Correspondence to Solen Rocher.

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Supplementary File 1 Sequences of forward and reverse SRAP primers used for SRAP-cDNA amplifications (DOCX 18 kb)

Supplementary File 2 Sequences obtained from sequencing of SRAP-cDNA amplicons. Fragment number corresponds to transcript-derived fragment (TDF) listed in Table 1 “fragment ID” column. Consensus (“contig”) sequences are provided when available. Forward or reverse sequences only are provided when one of both strands yielded sequences of low quality (TXT 25 kb)

Supplementary File 3 List of forward and reverse primers used for RT-qPCR amplification of SRAP-cDNA fragments identified in Table 1 and Additional File 4 and for the amplification of two reference genes (DOCX 15 kb)

Supplementary File 4 Results of electrophoresis of SRAP-cDNA fragments amplified with bulked cDNA samples from 50 plants either non-acclimated (NA, 0 h) or cold-acclimated (A) for 8 h at 2 °C and from 50 plants hardened two additional weeks at the non-lethal freezing temperature of − 2 °C (HF). PCR amplification of samples from the alfalfa cultivar Apica (ATF0) and population ATF5 obtained after five cycles of recurrent selection for freezing tolerance within that initial cultivar were performed with SRAP primers listed in Supplementary File 1. Numbers to the left and right of the gels refer to transcript-derived fragments (TDF) ID and corresponds to fragment ID in Table 1 and Supplementary File 2. Molecular weight ladder (MW) and functional annotations of each TDF based on sequence homology with M. truncatula reference genome are included in each gel (PPTX 6362 kb)

Supplementary File 5 Relative levels of expression of 23 SRAP-cDNA transcript-derived fragments (TDF) differentially expressed between crowns of alfalfa non-acclimated (0 h) and crowns cold-acclimated 8 h, 1 d, 3 d, 7 d, 15 d at 2 °C or hardened two additional weeks at the non-lethal freezing temperature of − 2 °C (HF). RT-qPCR amplifications were performed with samples from the alfalfa cultivars Apica (ATF0) and Evolution (ETF0) and populations ATF5 and ETF5 obtained after five cycles of recurrent selection for freezing tolerance in these two initial backgrounds. Means of five pots (10 plants pot). Gene expression was expressed relative to the mean of all samples within each population. Transcript-derived fragments (TDF) were grouped according to their expression profile as: A) Rapidly down regulated; B) Progressively down regulated; C) Transiently up regulated; D) Progressively up regulated. For each population, means with different letters are statistically different at P 0.05. Within each genetic background statistically significant response to recurrent selection at P 0.05, P 0.01 and P 0.001 are respectively indicated by *, ** and *** next to each acclimation treatment (XLSX 33 kb)

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Castonguay, Y., Rocher, S., Bertrand, A. et al. Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa. Euphytica 216, 27 (2020). https://doi.org/10.1007/s10681-020-2559-2

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  • Freezing tolerance
  • RT-qPCR
  • Recurrent selection
  • Bulk segregant analysis
  • Alfalfa